1. Field of the Invention
The present invention relates to an expression vector of a mud loach growth hormone gene and a fast-growing transgenic mud loach transformed with the expression vector thereof. More particularly, it relates to a cDNA gene encoding of a growth hormone which is isolated from a mud loach, an expression vector of a mud loach growth hormone gene containing a xcex2-actin gene regulation site of a mud loach, and a method of producing a mud loach of high growth rate by transforming it with the expression vector, and a fast-growing transgenic mud loach produced thereby.
2. Description of the Prior Art
A mud loach (Misgurnus mizolepis), which inhabits the northeastern Asian area including China, Taiwan, Japan, Russia, etc., is a representative fresh-water fish species in Korea and has been widely used as an excellent food and oriental medicine. Not long ago, the mud loach was profusely found in rice fields and rivers. However, due to an accelerated contamination of rivers and fields along with the abuse of pesticides, the amount of natural catch decreases every year. Considering that such a condition is not limited to only mud loaches, development of new fish culturing techniques as well as the improvement of fish breeds is required for an effective use and preservation of native fish resources. For this purpose, such research on the production of genetically improved fish through the recombination technology using a useful fish gene should be required.
The research for the production of a transformed fish by the recombinant gene began in the late 1970""s, in which a growth hormone gene was widely used. A growth hormone is a protein hormone whose structure and biological characteristics are similar with prolactin (PRL), chorionic somatomammotropin (CS; placental lactogen) and somato-lactin (SL). In all vertebrates, the growth hormone and PRL are produced in somatotroph and lactotroph of hypophysis, respectively, CS is produced in syncytotrophoblast of mammalian placenta, and SL in the pars intermedia cell of fish hypophysis. Synthesis and release of growth hormones are controlled by the GH releasing hormone (GRH) and GH release-inhibiting hormone (GIH; somatostatin). In case the glucose concentration in blood decreases, growth hormones are synthesized and released through the stimulation of GRH (Ecker, R., 1988, Chemical messengers and regulators, In: Animal physiology, 3rd ed. Freeman and company. pp. 266-328).
Growth hormones are expressed during a germ generation process (Pantaleon, M., E. J. Whiteside, M. B. Harvey, R. T.
Barnard, M. J. Waters, and P. L. Kaye, 1997, Proc. Natl. Acad. Sci. USA., 94: 5125-5130) and involved in all normal metabolism needed for the growth process, including tissue growth, especially stimulates growth of bones due to cartilage proliferation. Tissue growth is due, to the increase of the number of cells. The growth process through the growth hormone is achieved by stimulation of the production of growth promoting factors (somatomedins) such as IGF-1 not by the direct stimulation of cell growth. As biochemical functions of the growth hormone in living bodies occur, they increase the transportation of amino acids into muscle cells and protein synthesis. Further, they are involved in carbohydrate metabolism, that is, decreasing glucose utilization and increasing glucose synthesis in the liver, which is an antagonistic action to insulin. With relation to lipid metabolism, the growth hormone stimulates the release of fatty acids and glycerol from adipose tissue. Also, it relates to inorganic metabolism such as ion balance and stimulates cartilage formation and bone growth (Bentley, P. J., 1982, Comparative vertebrate endocrinology, 2nd ed. Cambridge Univ. Press, Cambridge. pp. 179-209; Murray, R. K., D. K. Granner, P. A. Mayers, and V. W. Rodwell, 1993, Pituitary and hypothalamic hormones, In: Harper""s Biochemistry. 23rd ed. Prentice-Hall Int., Inc. pp. 499-508).
Mammal growth hormone gene, whose size is about 2.5 kb, consists of five exons and four introns. Growth hormone genes of such fishes as rainbow trout, salmon and tilapia, whose size is about 4.5-5 kb, has six exons in which the 5th exon is divided by the 5th intron. Common carp have the same gene structure as humans and its gene size is about 3.5 kb. Tissue-specific expression of growth hormone and PRL is controlled by the transcription regulation protein, pit-1 and its upper transcription regulation factor, pit-1 binding site AA/TA/TTANCAT (SEQ ID NO:17) (Bodner, M., J. L. Castrillo, L. E. Theill, T. Deerinck, M. Ellisman, and M. Karin, 1988, Cell 55: 505-518). It is also known that the thyroid hormones T3, T4 and glucocorticoid are involved in the synthesis of the growth hormone in mammals (Evans, R. M., N. C. Birnberg, and M. G. Rosenfield, 1982, Proc. Natl. Acad. Sci. USA. 79: 7659-7663). However, such relations are not certain in fishes.
In early production of transformed fishes, mammal genes and their regulation sites are used. After a human growth hormone (hGH) was microinjected into goldfish (Zhu, Z., G. Liu, L. He, and S. Chen, 1985, Z. Angew. Ichthyol., 1: 31-43), it has been microinjected into many fishes including tilapia (Brem, G., B. Brenig, G. Horsgen-Schwark, and E. L. Winnacker, 1988, Aquaculture, 68: 209-219), rainbow trout (Chourrout, D., R. Guyomard, C. Leroux, F. Pourrain, and L. M. Houdebine, 1988, J. Cell Biochem. Suppl., 121: 188) and catfish (Dunham, R. A., J. Eash, J. Askins, and T. M. Towners, 1987, Trans. Am. Fish Soc., 116: 87-91). Further, mouse growth hormone gene (Penman, D. J., A. J. Beeching, S. Penn, and N. Maclean, 1990, Aquaculture, 85: 35-50) and bovine growth hormone gene (Gross, M. L., J. F. Schneider, N. Moav, C. Alvarez, S. Myster, Z. Liu, C. L. Hew, E. M. Hallerman, P. B. Hackett, K. S. Guise, A. J. Faras, and A. R. Kapuscinski, 1991, Aquaculture, 85: 115-128) are also used in microinjecting into fish genes.
In the experiments for producing transformed fish, such transgenes genes may be expressed and cause physiological changes. In some cases, however, such genes may be expressed without causing any physiological changes, or cannot be expressed at all. It is explained that non-expression is due to the fact that the gene regulation site of mammals cannot be recognized in fishes. Further, the reason that a specific hetero-gene cannot cause any physiological change despite its expression is that substrate-specific protein-protein interaction does not occur in cells. For example, growth hormone can effect its action through its binding with the growth hormone receptor in the surface of the cell wall. Likewise, in order that mammal growth hormone may be expressed in fishes and effect its action, interaction of mammal growth hormone with the fish growth hormone receptor must occur. Therefore, for effectiveness, the expression product of the gene to be transferred into fishes should have a structural similarity with that of fishes.
Among the successful cases of transfer of mammal growth hormone genes into fish cells, only few cases induced the increase of their growth rate, which shows the limitation of the expression of mammal genes in fish cells. Therefore, it is suggested that for an effective gene expression in fishes, genes and their regulation site should be selected from fishes, especially those of close classes. Therefore, for the improvement of a fish breed through gene recombination, cloning of fish-specific promoters and structural genes, along with the development of expression vectors used thereof is required.
After the growth hormone gene cDNA of rainbow trout was cloned and its sequence was identified (Agellon, L. B., and T. T. Chen, 1986, DNA, 5: 463-471), the growth hormone gene cDNA and genome clone of several fishes including common carp have been separated. Zhang et al. (Zhang, P., M. Hyat, C. Joyce, L. I. Gonzalez-Villasenor, C. M. Lin, R. A. Dunham, T. T. Chen, and D. A. Powers, 1990, Mol. Rep. Dev., 25: 3-13) reported that growth hormone can be expressed in the population F1 of common carp transformed with the growth hormone gene cDNA of rainbow trout.
In transmitting foreign genes to fish cells in culture or embryo in generation, the limitation factor is the effectiveness of the vector. After using a mammal-originated vector in the early days, it was reported that several promoters of some vertebrates or viruses can efficiently transmit foreign genes to fish cells (Foster, R., P. E. Olson, and M. Zaffarullah, 1990, Regulation of rainbow trout metallothionein genes. In: Transgenic models in Medicine and Agriculture. Church, R. (ed.) Willy-Liss, pp. 101-108; Inoue, K., S. Yamashita, J. Hata, S. Kabeno, S. Asada, E. Nagahisa, and T. Fujita, 1990, Cell Differ. Dev., 29: 123-128; Liu, Z., B. Moav, A. J. Faras, K. S. Guise, A. R. Kapuscinski, and P. B. Hackett, 1990, Bio/Technol., 8: 1268-1272; Friedenreich, H., and M. Schartl, 1990, Nucl. Acids Res., 18: 3299-3305; Bearzotti, M., E. Perrot, C. Michard-Vanhee, G. Jolivet, J. Attal, M. C. Theron, C. Puissant, M. Drano, J. J. Kopchick, R. Powell, F. Gannon, L. M. Houdebine, and D. Chourrot, 1992, J. Biotechnol., 26: 315-325). Zhu, et al. (Zhu, J., K. Xu, G. Li, Y. Xie, and L. He, 1986, Tongbao, 31: 988-990) succeeded in producing a transformed fish three times bigger than normal fish by microinjecting a human growth hormone gene bound in mouse metallothioneine (MT) promoter into mud loach, gold fish, carp, etc. Using a vertebrate and virus promoter in fishes, however, has such problems as a limited transcription factor in expression and consumers avoidance (Du, S. J., Z. Gong, C. L. Hew, C. H. Tan, and G. L. Fletcher, 1992, Mol. Mar. Biol. Biotechnol., 1(4/5): 290-300).
Considering the above, fish-originated promoters and genes are ideal for producing a transformed fish. Accordingly, promoters of the protamine gene of salmon (Jankowski, J. M., and G. H. Dixon, 1987, Biosci. Rep., 7: 955-963), metallothionein B gene of rainbow trout (Zafarullah, M., K. Bonham, and L. Gedamu, 1988, Mol. Cell. Biol., 8: 4469-4476), xcex2-actin gene of common carp (Liu, Z., B. Moav, A. J. Faras, K. Guise, A. R. Kapuscinski, and P. B. Hackett, 1990, Mol. Cell. Biol., 10: 3432-3440), antifreeze protein (AFP) of flatfish (Gong, Z., C. L. Hew, and J. R. Biekind, 1991, Mol. Mar. Biol. Biotechnol., 1: 64-72), etc. are cloned and used as regulation sites in producing an expression vector for fishes.
Among these promoters, MT gene promoters can show strong expression through the treatment of toxic substances, heavy metals, glucocorticoid, etc. However, such treating materials sometimes have toxicity or induce cancer during their metabolic processes, especially heavy metals are deposited in fish bodies and transported into final consumers (Liu, Z., B. Moav, A. J. Faras, K. Guise, A. R. Kapuscinski, and P. B. Hackett, 1990, Bio/Technol., 8: 1268-1272).
The actin gene, which is a cell structural protein gene in eukaryote, is often used as a regulation site for a stable and continuous expression of foreign genes. In vertebrates, actin has at least six isoforms showing some differences in amino acid sequences, whose expressions are controlled at various sites in generation. While xcex1-actin shows muscular specific expression, xcex2-actin gene promoters can express in all the non-muscular cells including muscular cells. Therefore, it is expected that xcex2-actin gene promoter bound in foreign genes can induce the expression of the foreign genes (Quitschke, W. W., Z. Y. Lin, L. D. P.-Zilli, and B. M. Paterson, 1989, J. Biol. Chem. 264(16): 9539-9546).
When the xcex2-actin gene regulation site is used in producing an expression vector for fishes, (1) it can induce expression in all tissues, (2) its expression can be induced and controlled at various steps, and (3) it can function in all fishes due to its similarity of nucleic acid sequences in species (Liu, Z., B. Moav, A. J. Faras, K. Guise, A. R. Kapuscinski, and P. B. Hackett, 1990, Mol. Cell. Biol., 10: 3432-3440).
It is an object of the present invention to provide a gene regulation site originated from fishes, and an expression vector including the gene regulation site, in order to induce expression of foreign genes in fishes.
It is another object of the present invention to provide a growth hormone expression vector for stimulating the growth of useful fishes as food or medicine, a mud loach transformed with the expression vector, and method of producing a fast-growing transgenic mud loach.
To achieve the object of the present invention, there is provided a DNA including xcex2-actin gene and xcex2-actin gene regulation site of mud loach, expressed as SEQ ID NO: 1.
In accordance with a further aspect of the present invention, there are provided an expression vector including xcex2-actin gene regulation site of mud loach, and a transformant including the expression vector.
To achieve another object of the present invention, there is provided a DNA including a growth hormone gene of a mud loach, expressed as SEQ ID NO: 8.
In accordance with a further aspect of the present invention, there is provided an expression vector, including a growth hormone gene of mud loach and xcex2-actin gene regulation site of mud loach.
In accordance with still another aspect of the present invention, there are provided a method of producing a mud loach of a high growth rate comprising the step of micro-injection of the expression vector into a fertilized egg, and a mud loach of a high growth rate transformed with the expression vector.